CPTAC Breast Cancer Tissue Proteomics

The xenograft tumor samples acquired from CPTAC program were generated from primary or metastatic breast tumors.^{11 (link)–14 (link)} The 50 mg tissue of each tumor sample was lysed with sonication in 8 M urea and 1 M NH₄HCO₃ pH 8, containing 75 mM NaCl. Inhibitors of phosphatase and O-GlcNAcase were added in the lysis buffer. After lysis, proteins were reduced with 5 mM DTT, alkylated with 10 mM IAA, and digested with LysC and trypsin (Promega) at 37 °C. The digested peptides were desalted on C18 SepPak columns (Waters). 400 μg of desalted peptides were then labeled by an individual channel of TMT10plex (Thermo Fisher Scientific). After TMT label reaction, all ten channels were combined and desalted on a C18 SepPak column. Basic reversed phase fractionation was performed on Agilent 1100 HPLC analytical system, generating 24 fractions for global proteome analysis and 13 combined fractions for phosphopeptide enrichment. Phosphopeptides were enriched from each of the 13 fractions using IMAC method and desalted by stage-tips. The global and phosphoproteomic study of CPTAC breast cancer tissues was described in a previous publication.^{11 (link)}